Salmonella enterica represents a considerable burden to human and animal health worldwide. A significant effort has been made to understand the pathogenesis of this intracellular pathogen and the host factors that mediate host defense. Clinical and experimental evidence have unequivocally demonstrated that CD4+ T cells and IFN? are critical for preventing systemic disease by non-typhoidal Salmonella. IFN? likely exerts diverse functions in resistance to this intracellular bacterium, including the activation of the antimicrobial arsenal of macrophages. Recent studies have indicated that IFN? synergizes with Salmonella ligands to enhance the transcription of iNOS. The resultant high NO synthesis mediates most of the profound and long-lasting anti-Salmonella activity of IFN?-primed macrophages. The molecular mechanism(s) by which IFN?- activated NO synthesis enhances the anti-Salmonella activity of macrophages remains, however, largely unknown. Most of the biological chemistry of NO stems from its interaction with metals in cytochromes of the electron transport chain, zinc fingers of DNA-binding metalloproteins and iron-sulfur clusters of dehydratases. Despite their abundance, NO-related inhibition of these metal centers is secondary in order of importance to its effects on SPI2 function. The goal of this application is to identify the molecular mechanisms underlying the RNS-mediated repression of SPI2 transcription. It is hypothesized that NO congeners repress SPI2 transcription by S-nitrosylating (-SNO) C203 of the dimerization domain of the SsrB response regulator. Specifically, we propose to 1) characterize the RNS-mediated modifications that inactivate SsrB regulatory functions; 2) examine SPI2 function in the context of Salmonella antinitrosative defenses; 3) select for ssrB variant alleles that render SsrB signaling insensitive to RNS; and 4) characterize SsrB residues critical for dimerization.